CN113913495B - Duplex UMI adaptor and sequencing method - Google Patents
Duplex UMI adaptor and sequencing method Download PDFInfo
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Abstract
The invention relates to the field of biotechnology, in particular to Duplex UMI connectors and a sequencing method. The design of the fixed Duplex UMI joint can correct UMI errors in the process of letter generation analysis, and improves the accuracy of original DNA molecule counting; in addition, a set of rapid quality inspection method is established for the joint, and the hybridization step can be omitted, so that the detection period is greatly shortened.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to Duplex UMI connectors and a sequencing method.
Background
ctDNA is free DNA (cfDNA) released by tumor cells after necrosis and apoptosis, has short half-life in blood, and can reflect dynamic change of tumor in real time. Current techniques for ctDNA liquid biopsy are mainly ARMS-PCR, digital PCR (ddPCR) and second generation sequencing (NGS). NGS is the most widely used gene detection technology that can detect multiple different variants of multiple genes simultaneously. However, because the NGS experimental process is relatively complex, some amplification and sequencing errors are inevitably introduced in the library construction, target region capturing and sequencing processes, and are called background noise, ctDNA detection tends to have low mutation frequency and is greatly interfered by the background noise, and low-frequency mutation from ctDNA samples tends to be submerged in the background noise, so that false negative or false positive results are caused, which limits the sensitivity and specificity of ctDNA detection.
In order to improve the sensitivity and specificity of ctDNA detection, a linker, duplex UMI (Duplex Unique Molecular indentifier), was designed in the primer for detection. DuplexUMI can correct for false bases that randomly occur during PCR or sequencing, and only retains the actual mutation in the sample during the belief analysis. However, most of Duplex UMI sequences on the market at present are composed of random sequences, so that the accuracy of the synthesis of manufacturers cannot be evaluated; when the manufacturer purifies, two UMIs are possibly polluted by each other due to a purification column, but the synthesis mode of the random sequence cannot be distinguished; the sequencing error rate of the sequencer of either the Illumina or MGI platform is about 0.1%, and the random sequence synthesis mode cannot identify the reads with the sequencing errors; in addition, the random sequence synthesis has the problems of low continuous repeated base synthesis efficiency and low sequencing quality value.
Disclosure of Invention
In view of the above, the present invention aims to provide Duplex UMI adapter and sequencing method for further improving detection accuracy.
The Duplex UMI connectors provided by the invention are 64 connectors in each group, and each connector keeps base balance;
continuously repeating less than or equal to 2 bases in each joint sequence, wherein the editing distance of any two joint sequences is more than or equal to 2;
each group comprises:
16 linkers of 6nt length, AG or AC at the 3' -terminal base,
16 linkers of 6nt length, the 3' -terminal base of which is TG or TC,
16 linkers of 5nt length with a 3' terminal base of C
16 linkers of 5nt length with G at the 3' terminal base.
In Duplex UMI linkers provided by the invention, the nucleic acid sequences of each group of 64 linkers are respectively: CTAGTG, AAGATC, TGCCAG, GCCAAC, ATCCTG, GTGCTG, GCTATC, ACTAAC, AGTATC, CCTCAG, CGGTAC, TGATAC, TCTGTG, AGCAAC, GGAATC, TCCTTC, GTACAG, TCAGAG, GCTGAG, CGCGTG, GAGCAG, CTGTTC, TGAGTG, TTGCAG, GACCTG, GCCGTG, CCGAAC, TAACTG, AACCAG, TATTAC, AACGTG, CACTAC, GACTG, CTGGC, TGTGG, TCAAC, TTCTG, GCAAG, TGTAC, GCTTC, AGATG, TTACC, CGTTG, CCACG, TTAAG, AATTG, AGCCG, AAGCG, GATAG, CTATC, CAAGC, CTTAG, ACGGC, ATGCC, ATTGG, TTGTC, CACAC, CGACC, GACGC, TATGC, TGGCG, GGCAC, GTGAG and TATCG.
The invention also provides a Genecast UMI linker comprising: 64 connectors 1 and 64 connectors 2,
the structure of the joint 1 is a frame 1- (N) xT; the structure of the joint 2 is (N) x-frame 2;
(N) x is a Duplex UMI adapter according to the present invention.
In an embodiment of the present invention,
the sequence of frame 1 is:
5’-GTCTTCCTAAGACCGCTTGGCCTCCGACTT-3’,
or 5'-TTGTCTTCCTAAGACCGCTTGGCCTCCGACTT-3';
the sequence of frame 2 is:
5’- AAGTCGGATCGTAGCCATGTCGTTCTGTGAGCCAAGGAGT-3’，
or 5'-AAGTCGGATCGTAGCCATGTCGTTCTGTGAGCCAAGGAGTTG-3'.
In the preparation method of the Genecast UMI joint,
the preparation system comprises: frame 1, frame 2, any one of Duplex UMI connectors described herein, annealing buffer, EB buffer;
the annealing buffer solution comprises water, 50 mmol/L Tris-Cl, 5 mmol/L EDTA and 250 mmol/L NaCl;
the preparation process comprises the following steps:
the temperature of the hot cover is 105 ℃, the temperature of the hot cover is kept at 95 ℃ for 4min,
cooling to 90 ℃ at 0.1 ℃/s, and keeping at 90 ℃ for 2min;
cooling to 85 ℃ at 0.1 ℃/s, and keeping at 85 ℃ for 2min;
cooling to 80 ℃ at 0.1 ℃/s, and keeping at 80 ℃ for 2min;
cooling to 75 ℃ at 0.1 ℃/s, and keeping at 75 ℃ for 2min;
cooling to 70 ℃ at 0.1 ℃/s, and keeping at 70 ℃ for 2min;
cooling to 65 ℃ at 0.1 ℃/s, and keeping at 65 ℃ for 2min;
cooling to 60 ℃ at 0.1 ℃/s, and keeping at 60 ℃ for 2min;
cooling to 55 ℃ at 0.1 ℃/s, and keeping at 55 ℃ for 2min;
cooling to 50 ℃ at 0.1 ℃/s, and keeping at 50 ℃ for 2min;
cooling to 45 ℃ at 0.1 ℃/s, and keeping at 45 ℃ for 2min;
cooling to 40 ℃ at 0.1 ℃/s, and keeping at 40 ℃ for 2min;
cooling to 35 ℃ at 0.1 ℃/s, and keeping the temperature at 35 ℃ for 2min;
cooling to 30 ℃ at 0.1 ℃/s, and keeping at 30 ℃ for 2min;
cooling to 25 ℃ at 0.1 ℃/s, and keeping at 25 ℃ for 2min;
preserving at 4 ℃.
The Genecast UMI linker of the invention is used in the construction of sequencing libraries.
The invention also provides a construction method of the sequencing library, which constructs the library by using the Genecast UMI joint.
The construction method in some embodiments comprises:
and (3) connecting ctDNA with the linker after adding A for end repair, constructing a library, and purifying the obtained product to obtain the library.
In the invention, after the library is constructed, the steps of hybridization, elution and library amplification are also included.
The invention also provides a sequencing method, which is used for detecting the library obtained by the construction method on the machine.
The design of the fixed Duplex UMI joint can correct UMI errors in the process of letter generation analysis, and improves the accuracy of original DNA molecule counting; in addition, a set of rapid quality inspection method is established for the joint, and the hybridization step can be omitted, so that the detection period is greatly shortened.
Drawings
Fig. 1 shows a general AT connection schematic;
FIG. 2 shows a CG correction design for GenecastDuplexUMI;
FIG. 3 shows a 2100 plot of the GenecastDuplexUMI preparation;
FIG. 4 shows a 2100 plot of DuplexUMI preparation of CN 113005121A;
FIG. 5 shows a 2100 plot of DuplexUMI preparation of CN 109988819A;
FIG. 6 shows a duplex reads data comparison after detection of two clinical human CtDNA samples by three linkers;
FIG. 7 shows a comparison of simplex reads data after detection of two clinical human CtDNA samples by three linkers;
FIG. 8 shows a single reads data comparison after detection of two clinical human CtDNA samples by three linkers;
FIG. 9 shows a comparison of dcsV data after detection of two clinical human CtDNA samples by three linkers;
FIG. 10 shows the ratio of 64 UMIs in the result to the data amount after the test of example 2;
FIG. 11 shows a comparison of the detection times of example 3 and example 4;
fig. 12 shows comparison of the detection results of example 3 and example 4.
Detailed Description
The invention provides Duplex UMI linkers and sequencing methods, and those skilled in the art can suitably modify the process parameters to practice, given the teachings herein. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For definitions and terms in the art, the expert may refer specifically to Current Protocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are standard 3-letter and/or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.
The principle of the sequencing is shown in Newman A M, lovejoy A F, kgrass D M, et al Integrated digital error suppression for improved detection of circulating tumor DNA [ J ]. Nature biotechnology, 2016, 34 (5): 547-555. To improve the accuracy and sensitivity of the sequencing, the adaptor of the invention is added at both ends of the adaptor used for sequencing. The DuplexUMI linker of the present invention is different from the random linker used in the prior art, and can effectively avoid errors introduced in the sequence synthesis process, the PCR amplification process or the sequencing process of a sequencer. Therefore, nonspecific detection caused by random linkers can be avoided, and false positive reporting is reduced.
Furthermore, previously, the ligation of Duplex UMI and insert was mostly performed by AT ligation, and a few by blunt-end ligation. As shown in FIG. 1, when the connection product is subjected to PCR enrichment and then is sequenced, the base AT the position is A/T when the connection position of the AT is detected, and the base imbalance is caused. Extreme base unbalance can lead to reduced sequencing quality at this position, which in turn affects QC results for the whole reads. In order to solve the problem, C or G is additionally added after 32 joints with the end base A or T AT the 3' end, as shown in figure 2, when the connection product is subjected to PCR enrichment and then is sequenced, the base AT the position is A/T/C/G and is balanced when the AT connection position is detected.
In the present invention, the base balance means that the number of A, T, C, G bases in one linker position is similar, for example, A, T, C, G bases account for 21.9%, 29.7%, 23.4% and 25.0% in the 64-group linker 1, respectively, at the first base position of the linker. The specific table is as follows:
the Genecast UMI linker of the present invention is a linker used for constructing a library, wherein the framework part comprises a framework 1 and a framework 2. In the construction process of the library, various optional skeletons can be selected to construct the linker. In the embodiment of the invention, the design of the framework sequences can be various:
the sequence of frame 1 is:
5’-TTGTCTTCCTAAGACCGCTTGGCCTCCGACTT-3’
or a combination of 5'-GTCTTCCTAAGACCGCTTGGCCTCCGACTT-3',
wherein 5'-TTGTCTTCCTAAGACCGCTTGGCCTCCGACTT-3' is the framework sequence used in Table 3, which is identical to universal linker sequence 1 in the CN202110450556.0 patent;
the sequence of frame 2 is:
5'-AAGTCGGATCGTAGCCATGTCGTTCTGTGAGCCAAGGAGTTG-3' or 5'-AAGTCGGATCGTAGCCATGTCGTTCTGTGAGCCAAGGAGT-3' of the number of the pins,
wherein 5'-AAGTCGGATCGTAGCCATGTCGTTCTGTGAGCCAAGGAGTTG-3' is the framework sequence used in table 3;
the construction method of the Genecast UMI joint is to connect the framework with the Duplex UMI joint.
The system for preparing the Genecast UMI linker comprises any one of a frame 1, a frame 2 and a Genecast UMI linker, an annealing buffer and an EB buffer. In a total of 64 reaction systems were constructed in which frame 1 and frame 2 were annealed together to finally form 64 Genecast UMI linkers prepared.
The library of the invention is a library for second generation sequencing. In the library construction process, samples can be subjected to end repair and linker ligation, and sequencing libraries can be obtained only after library construction. The product may also be purified, sorted, hybridized, eluted after PER PCR, and then subjected to library amplification.
The test materials adopted by the invention are all common commercial products and can be purchased in the market. The invention is further illustrated by the following examples:
example 1 Genecast UMI linker preparation
1.1 5nt UMI design and screening was performed according to the following principles: (1) requiring continuous repetition of less than or equal to 2 bases; (2) the editing distance of any two sequences is required to be more than or equal to 2; (3) 64 sequences in each group, wherein the last base of 32 sequences is A or T, and the last base of the other 32 sequences is G or C; (4) each position of 5nt is base balanced, i.e., the A/C/T/G ratio should be 25% + -5%. The design results are shown in Table 1.
TABLE 15 nt UMI design results
1.2 to solve the problem of base imbalance caused by AT ligation, C/G is additionally added after the last 32 sequences with bases A or T, design principle: (5) the sequence complementary to the 3' end is added with the same C or G. The design results after this treatment are shown in Table 2.
TABLE 2 design results of 5nt UMI after base Balancing
1.3 According to the sequence characteristics of the MGI sequencing platform, we will here:
the universal framework sequence 1 is designed as:
GTCTTCCTAAGACCGCTTGGCCTCCGACTT（SEQ ID NO:1）-(N)xT，
the generic framework sequence 2 is designed as:
(N)x-AAGTCGGATCGTAGCCATGTCGTTCTGTGAGCCAAGGAGT（SEQ ID NO:2）。
where x=5 or 6.
And assembling the UMI sequence and the frame sequence to obtain the synthesized Genecast UMI sequence.
Table 3 Genecast UMI linker sequence for one of the frameworks
1.4 Duplex UMI preparation
The 5×annealing buffer is configured as follows:
table 45 annealing buffer formulation
The joint annealing system was prepared according to the following reaction system:
TABLE 5 Joint annealing reaction System
The adaptor annealing was performed by PCR following the following procedure:
TABLE 6 annealing procedure
The 64 annealed adaptors were mixed in equal amounts.
Comparative example 1
Synthetic linkers are described in patent CN113005121 a. The length of the molecular tag is 6-12 bp, preferably 8-10 bp; preferably the molecular tag is a sequence of A, T, G and C base compositions synthesized randomly.
Comparative example 2
Synthetic linkers are described according to the summary of patent CN109988819 a. It comprises: a core sequence; the random sequence (N) N, N being the random sequence, is N in total.
Example 2 human CtDNA detection
Taking ctDNA (obtained by extracting human plasma samples) for carrying out sampling detection and library establishment, wherein the detailed library establishment method comprises the following steps of:
1. end repair plus A
30ng of ctDNA was taken, the reaction enzyme and buffer were prepared in advance, the total volume was made 60. Mu.L by adding water, and after mixing, incubated in a PCR apparatus at 20℃for 30 minutes and at 65℃for 30 minutes. The reaction system is as follows:
2. ligation reaction
The following reagents were sequentially added to the mixture 1 after the end repair and addition of A, and after mixing, the mixture was incubated in a PCR apparatus at 20℃for 15 minutes.
3. Purifying after connection: the ligation product was purified using 0.8 Xmagnetic beads and dissolved with 22. Mu.L of water.
4. Library construction: the reagents were added sequentially as follows to prepare a mixed solution in a PCR tube.
Mix the liquid and cover the PCR tube, briefly centrifuge and collect the liquid.
Placing the prepared mixed solution into a PCR instrument, and amplifying according to the following reaction procedures:
5. library purification and sorting
The PrePCR product was purified with 0.5 x magnetic beads and dissolved with 100 μl of water.
6. Hybridization and elution
Hybridization and elution operations were performed using the capture Panel of IDT and wash reagent.
7. Library amplification
The mixed solution is prepared according to the following formula.
The mixture was mixed with the hybridization eluate, vortexed thoroughly, centrifuged briefly and the total volume was 50uL. Amplification was performed according to the following reaction procedure:
remarks: the number of cycles depends on the hybridization capture panel
8 library purification after capture: 1.2 Xthe beads were purified and dissolved in 22. Mu.L of water.
9. And (5) on-line and letter generation analysis: the library was subjected to MGI sequencing platform and analyzed using a letter flow.
Example 3 Phox DNA detection
The adaptor prepared in example 1 was used to detect the Phix bacterial DNA by the same method as in example 2. Phix bacterial DNA was simulated with the following sequence:
the sense strand sequence is:
CAAGCTAGAGTTCAACTGTCGTAACGCTATTCACTTCAACCTAGTGTGCGAA，
the antisense strand sequence is:
TCGCACACTAGGTTGAAGTGAATAGCGTTACGACAGTTGAACTCTAGCTTGA。
EXAMPLE 4 Phox DNA Rapid detection
In rapid detection for Duplex UMI, the step of hybridization capture may not be performed, thereby saving time.
1. Ligation reaction:
30ng of Phix DNA was taken, and after mixing, the following reagents were added in sequence and incubated in a PCR apparatus for 15 minutes at 20 ℃.
2. Post-ligation purification
The ligation product was purified using 0.8 Xmagnetic beads and dissolved with 22. Mu.L of water.
3 PrePCR
The reagents were added sequentially as follows to prepare a mixed solution in a PCR tube.
Mix the liquid and cover the PCR tube, briefly centrifuge and collect the liquid.
Placing the prepared mixed solution into a PCR instrument, and amplifying according to the following reaction procedures:
4. library purification and sorting
The PrePCR product was purified with 0.5 x magnetic beads and dissolved with 100 μl of water.
5 on-line and letter generation analysis
The library was subjected to MGI sequencing platform and analyzed using a letter flow.
The primer of the Phix sequence can lead the rapid detection method to break through the restriction of index non-collision, and can be put on the machine together with the library of the same index. And (3) taking the Phix bacterial sequence for data splitting, and splitting the rapidly detected data independently.
Effect analysis
1. Joint preparation efficiency detection
The joints prepared in example 1 and comparative examples 1-2 were identified, as shown in FIG. 3, genecast GenecastDuplexUMI, which had only a single main peak, demonstrated that the preparation efficiency was very high; fig. 4-5 show detection patterns of two random joints after construction in the prior art, and fig. 4 shows that the preparation efficiency is low and more joints which are not annealed successfully exist as main peaks of 4, especially 38 bp; FIG. 5 also illustrates that V is inefficient to produce and has a peak of 88bp at the highest, indicating that there may be overlap of the linkers.
2. The detection effects of the library obtained by constructing the three joints of example 1 and comparative examples 1-2 were compared:
1.duplex data comparison
Statistical methods for duplex reads, simplex reads and single reads are described with reference to FgBio (http:// fulcramgenomics. The same sample is subjected to warehouse building/loading by using the joint of the embodiment 1, the comparison example 1 and the comparison example 2 respectively, then the data of the next machine is intercepted and analyzed, and the analysis results are shown in fig. 6-8. As shown in FIG. 6, the duplex reads data for the example 1 linker far exceeds the linkers of comparative examples 1-2. As shown in FIG. 7, the simplex reads data for the example 1 linker is lower than for the comparative examples 1-2 linkers. As shown in FIG. 8, the single reads data for the example 1 linker is lower than for the comparative examples 1-2.
2. dcsV data comparison
dcsV refers to mutations supported by DNA duplex. The higher dcsV indicates better Duplex UMI performance.
dcsV statistical methods refer to Vardict (https:// gitsub. Com/AstraZeneca-NGS/VarDict) or samtools (http:// www.htslib.org /).
The results are shown in FIG. 9, and the results show that the structure of example 1 has stronger performance than comparative examples 1-2.
3. The properties of the joint obtained in example 1 were examined
1. The statistics of the proportion of the adaptor prepared in example 1 to the data amount in the sequencing is shown in fig. 10, and the results show that the data of 64 groups of UMIs account for 99.53% of the total data amount, which indicates that the accuracy of the adaptor sequencing obtained data is high.
2. The results obtained by library construction, capturing and on-line detection of the joint prepared in example 1 in example 2 were subjected to a letter generation analysis, and the results are shown in Table 7:
TABLE 7 results of raw letter analysis of clinical samples
The sample was subjected to digital PCR assay with the following assay results:
TABLE 8 digital PCR set values for clinical samples
The results show that the Genecast UMI linker prepared in example 1 can detect 0.05% of SNV/Indel mutations.
4. Comparison of library construction method of example 3 and library construction method of example 4
Comparing the time period used for the library construction method of example 3 with that of example 4, the result is shown in FIG. 11, and the library construction method of example 4 greatly shortens the experimental operation time compared with that of example 3. The results of the analysis were compared, and the results are shown in FIG. 12, in which the sequencing results of example 3 and example 4 are identical. The library construction method of example 4 is shorter than the method of example 3 and maintains good sequencing results, so that the adaptor provided by the invention can be used for rapid quality inspection of Duplex UMI without the capturing step.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Sequence listing
<110> zhen and (Beijing) biotechnology limited; tin-free fine and biological technology Co.Ltd
<120> Duplex UMI linker and sequencing method
<130> MP21021371
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 30
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
gtcttcctaa gaccgcttgg cctccgactt 30
<210> 2
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
aagtcggatc gtagccatgt cgttctgtga gccaaggagt 40
Claims (9)
- Duplex UMI linker, characterized in that the nucleic acid sequences of 64 linkers of each group are: CTAGTG, AAGATC, TGCCAG, GCCAAC, ATCCTG, GTGCTG, GCTATC, ACTAAC, AGTATC, CCTCAG, CGGTAC, TGATAC, TCTGTG, AGCAAC, GGAATC, TCCTTC, GTACAG, TCAGAG, GCTGAG, CGCGTG, GAGCAG, CTGTTC, TGAGTG, TTGCAG, GACCTG, GCCGTG, CCGAAC, TAACTG, AACCAG, TATTAC, AACGTG, CACTAC, GACTG, CTGGC, TGTGG, TCAAC, TTCTG, GCAAG, TGTAC, GCTTC, AGATG, TTACC, CGTTG, CCACG, TTAAG, AATTG, AGCCG, AAGCG, GATAG, CTATC, CAAGC, CTTAG, ACGGC, ATGCC, ATTGG, TTGTC, CACAC, CGACC, GACGC, TATGC, TGGCG, GGCAC, GTGAG and TATCG.
- A genecast UMI adapter comprising: 64 connectors 1 and 64 connectors 2,the structure of the joint 1 is a frame 1- (N) xT; the structure of the joint 2 is (N) x-frame 2;(N) x is a Duplex UMI linker as described in claim 1.
- 3. The Genecast UMI adapter of claim 2, wherein the adapter is configured to,the sequence of frame 1 is:5’-GTCTTCCTAAGACCGCTTGGCCTCCGACTT-3’,or 5'-TTGTCTTCCTAAGACCGCTTGGCCTCCGACTT-3';the sequence of frame 2 is:5’- AAGTCGGATCGTAGCCATGTCGTTCTGTGAGCCAAGGAGT-3’，or 5'-AAGTCGGATCGTAGCCATGTCGTTCTGTGAGCCAAGGAGTTG-3'.
- 4. A method for producing a Genecast UMI adapter according to claim 2, characterized in that,the preparation system comprises: frame 1, frame 2, duplex UMI adapter of claim 1, annealing buffer, EB buffer;the annealing buffer solution comprises water, 50 mmol/L Tris-Cl, 5 mmol/L EDTA and 250 mmol/L NaCl;the preparation process comprises the following steps:the temperature of the hot cover is 105 ℃, the temperature of the hot cover is kept at 95 ℃ for 4min,cooling to 90 ℃ at 0.1 ℃/s, and keeping at 90 ℃ for 2min;cooling to 85 ℃ at 0.1 ℃/s, and keeping at 85 ℃ for 2min;cooling to 80 ℃ at 0.1 ℃/s, and keeping at 80 ℃ for 2min;cooling to 75 ℃ at 0.1 ℃/s, and keeping at 75 ℃ for 2min;cooling to 70 ℃ at 0.1 ℃/s, and keeping at 70 ℃ for 2min;cooling to 65 ℃ at 0.1 ℃/s, and keeping at 65 ℃ for 2min;cooling to 60 ℃ at 0.1 ℃/s, and keeping at 60 ℃ for 2min;cooling to 55 ℃ at 0.1 ℃/s, and keeping at 55 ℃ for 2min;cooling to 50 ℃ at 0.1 ℃/s, and keeping at 50 ℃ for 2min;cooling to 45 ℃ at 0.1 ℃/s, and keeping at 45 ℃ for 2min;cooling to 40 ℃ at 0.1 ℃/s, and keeping at 40 ℃ for 2min;cooling to 35 ℃ at 0.1 ℃/s, and keeping the temperature at 35 ℃ for 2min;cooling to 30 ℃ at 0.1 ℃/s, and keeping at 30 ℃ for 2min;cooling to 25 ℃ at 0.1 ℃/s, and keeping at 25 ℃ for 2min;preserving at 4 ℃.
- 5. Use of the Genecast UMI linker of claim 2 or 3 in sequencing library construction.
- 6. A method for constructing a sequencing library, comprising constructing a library using the Genecast UMI linker according to claim 2 or 3.
- 7. The method of construction according to claim 6, comprising:the ctDNA is subjected to end repair and a, then is connected with the linker of claim 2 or 3, then library construction is performed, and the obtained product is purified to obtain a library.
- 8. The method of claim 7, further comprising the steps of hybridization, elution, and library amplification after the library is constructed.
- 9. A sequencing method, characterized in that the library obtained by the construction method according to any one of claims 6 to 8 is detected on-machine.
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| CN113005121A (en) * | 2021-04-25 | 2021-06-22 | 纳昂达(南京)生物科技有限公司 | Linker elements, kits and uses related thereto |
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| CN113005121A (en) * | 2021-04-25 | 2021-06-22 | 纳昂达(南京)生物科技有限公司 | Linker elements, kits and uses related thereto |
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